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Rhododendron Album Blume Inhibits Inos and COX-2 Expression in LPS-Stimulated RAW264.7 Cells Through the Downregulation of NF-Κb Signaling

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Rhododendron Album Blume Inhibits Inos and COX-2 Expression in LPS-Stimulated RAW264.7 Cells Through the Downregulation of NF-Κb Signaling INTERNATIONAL JOURNAL OF MOLECULAR MEDICINE 35: 987-994, 2015 Rhododendron album Blume inhibits iNOS and COX-2 expression in LPS-stimulated RAW264.7 cells through the downregulation of NF-κB signaling JI-WON PARK1,2, OK-KYOUNG KWON1,3, JUNG-HEE KIM1, SEI-RYANG OH1, JAE-HONG KIM2, JIN-HYUB PAIK4, BAMBANG MARWOTO5, RIFATUL WIDJHATI5, FIFIT JUNIARTI5, DODDY IRAWAN5 and KYUNG-SEOP AHN1 1Natural Medicine Research Center, Korea Research Institute of Bioscience and Biotechnology, Chungbuk 363-883; 2College of Life Sciences and Biotechnology, Korea University, Seoul 136-701; 3Department of Toxicology, College of Pharmacy, Chungnam National University, Daejeon 305-764; 4International Biological Material Research Center, Research Institute of Bioscience and Biotechnology, Daejeon 305-806, Republic of Korea; 5Center of Pharmaceutical and Medical Technology, The Agency for the Assessment and Application of Technology (BPPT), Kawasan Puspiptek Serpong, Tangerang, Banten 15314, Indonesia Received April 25, 2014; Accepted February 2, 2015 DOI: 10.3892/ijmm.2015.2107 Abstract. Rhododendron album Blume (RA) has traditionally the suppression of NF-κB activation. We therefore suggest that been used as an herbal medicine and is considered to have RAME may be prove to be an effective therapeutic agent for the anti-inflammatory properties. In the present study, we screened treatment of inflammatory diseases. RA extracts with anti-inflammatory properties. The biological effects of an RA methanol extract (RAME) on inflammation Introduction were investigated in lipopolysaccharide (LPS)-stimulated mouse RAW264.7 cells. We investigated the effects of RAME on the Inflammation is a central feature of a number of pathophysi- production of nitric oxide (NO) and prostaglandin E2 (PGE2) ological conditions in response to tissue injury and in the host in LPS-stimulated RAW264.7 cells. To explore the anti-inflam- defense against invading microbes. Pro-inflammatory cells, matory mechanisms of RAME, we measured the mRNA and mainly activated macrophages, mediate most of the cellular protein expression of pro-inflammatory mediators induced by and molecular pathophysiology of inflammation by producing RAME in the LPS-stimulated RAW264.7 cells by RT-PCR and cytokines and other pro-inflammatory molecules, including western blot analysis, respectively. RAME significantly inhibited prostaglandins (PGs), enzymes and free radicals, such as the production of NO, PGE2, interleukin (IL)-6, IL-1β and tumor nitric oxide (NO) (1,2). NO, which is a strong stimulus, has necrosis factor (TNF)-α in the LPS-stimulated RAW264.7 cells. been implicated in the pathogenesis of a variety of inflamma- It also suppressed the mRNA and protein expression of inducible tory diseases and is synthesized from L-arginine by inducible NO synthase (iNOS), cyclooxygenase-2 (COX-2) and mitogen- NO synthase (iNOS) (3). Moreover, cyclooxygenase-2 (COX-2) activated protein kinases (MAPKs) with a concomitant decrease is also involved in the pathology of chronic inflammation. in the nuclear translocation of nuclear factor-κB (NF-κB) in COX-2 is an inducible enzyme and is induced by lipopolysac- the LPS-stimulated RAW264.7 cells. These results indicate charides (LPS) and several cytokines (4). Thus, iNOS and that RAME inhibits LPS-induced inflammatory responses. COX-2 are plausible targets for the prevention or treatment of These effects were considered to be strongly associated with chronic inflammatory disorders, and the development of effec- tive iNOS and COX-2 inhibitors represents a major advance in the treatment of inflammatory processes (5,6). The nuclear factor-κB (NF-κB) and mitogen-activated protein kinase (MAPK) signaling pathways are key regula- Correspondence to: Dr Kyung-Seop Ahn, Natural Medicine tors of the expression of inflammatory mediators, including Research Center, Korea Research Institute of Bioscience and Biotechnology, 30 Yeongudanji-ro, Ochang-eup, Cheongwon-gun, COX-2 and iNOS (7,8). In unstimulated cells, the dimeric Chungbuk 363-883, Republic of Korea form of NF-κB is trapped in the cytoplasm by physical asso- E-mail: [email protected] ciation with an inhibitory protein, IκB (9,10). A number of stimuli, including LPS, cytokines and viruses, activate NF-κB Key words: Rhododendron album Blume, inflammation, lipopoly- through several signal transduction pathways, leading to the saccharide, mitogen-activated protein kinases, nuclear factor-κB phosphorylation of IκB. The activation of MAPKs contributes to the production of inflammatory mediators, such as COX-2 988 PARK et al: ANTI-INFLAMMATORY EFFECTS OF RAME and iNOS in activated macrophages (11), and specific MAPK NO assay. The RAW264.7 cells were plated at a density of inhibitors suppress COX-2 and iNOS expression (12,13). 2.5x105 cells/ml in 96-well plates and then incubated with or Rhododendron album Blume (RA) is a species of plant without LPS (0.5 µg/ml) in the absence or presence of various that belongs to the Ericaceae family and it is endemic to concentrations of RAME for 24 h. The NO accumulation in Indonesia. It is mostly found in the increasingly smaller high the supernatant was assessed by a Griess reaction. Each 100 µl elevation forests throughout West and Central Java. RA is of the culture supernatant was mixed with an equal volume grown widely in nurseries throughout the world, particularly of Griess reagent [0.1% N-(1-naphthyl)-ethylenediamine, in the United States. RA, along with many other variations 1% sulfanilamide in 5% phosphoric acid] and incubated at and hybrids, is a popular plant in the landscaping industry and room temperature for 10 min. The absorbance was measured is used for a variety of decorations, including wedding and at 540 nm using a microplate reader, and a series of known special occasion bouquets, corsages and other floral arrange- concentrations of sodium nitrite was used as a standard. ments. However, to the best of our knowledge, there are no data available in the literature on the anti-inflammatory Prostaglandin E2 (PGE2) assay. The PGE2 concentration in activity of this plant. Therefore, the aim of the present study the supernatant was determined using a commercially avail- was to evaluate the anti-inflammatory activity of a methanol able PGE2 ELISA kit (Cayman Chemical Co., Ann Arbor, MI, extract of this plant. USA), according to the manufacturer's instructions. The PGE2 As part of our continuing search to identify anti-inflam- concentrations were determined by measuring the absorbance matory compounds from natural products, we identified at 405 nm. RA as a potent inhibitor of iNOS and COX-2 expression in LPS-stimulated RAW264.7 cells. To the best of our knowl- Reverse transcription (RT)-PCR. RT-PCR was performed for edge, in this study, we demonstrate for the first time that RA the detection of the mRNA expression of iNOS, COX-2, inter- is a novel natural anti-inflammatory compound that blocksthe leukin (IL)-6, IL-1β, tumor necrosis factor (TNF)-α and LPS-induced activation of the NF-κB and MAPK pathways. β-actin. Briefly, following stimulation of the RAW264.7 cells with LPS (0.5 µg/ml) of for 6 h, total RNA was isolated using Materials and methods TRIzol™ reagent (Invitrogen) as recommended by the manu- facturer. Subsequently, a reverse transcription reaction was Preparation of the RA extract. Plant material was collected carried out using a kit for producing cDNA (Qiagen GmbH, from Cantigi, Indonesia in 2008. The plant was identi- Hilden, Germany). PCR was carried out using specific forward fied by the botanist, Agung Sedayu. A voucher specimen and reverse primers and a premix according to the manufac- (KRIB 0019989) has been deposited in the herbarium of the turer's instructions (Bioneer Corp., Daejeon, Korea). The Korea Research Institute of Bioscience and Biotechnology. following conditions were used for each PCR reaction: 94˚C for The RA plant material was treated with methanol (MeOH) 5 min (1 cycle); 94˚C for 30 sec, 60˚C for 30 sec, and 72˚C for and sonicated several times at room temperature for 3 days to 45 sec (for 30 cycles); and a final extension phase at 72˚C for produce an extract. 10 min. The primer sequences for the analysis of mRNA expre- sion were as follows: iNOS, 5'-CAA GAG TTT GAC CAG Cell culture. RAW264.7 cells [American Type Culture AGG ACC-3' (sense) and 5'-TGG AAC CAC TCG TAC TTG Collection (ATCC); Manassas, VA, USA], a mouse monocyte/ GGA-3' (antisense); COX-2, 5'-GAA GTC TTT GGT CTG macrophage cell line, were maintained in a 95% air, 5% CO2 GTG CCT G-3' (sense) and 5'-GTC TGC TGG TTT GGA ATA atmosphere in Dulbecco's modified Eagle's medium (DMEM; GTT GC-3' (antisense); IL-1β, 5'-GTG TCT TTC CCG TGG Gibco-BRL, Grand Island, NY, USA) supplemented with ACC TT-3' (sense) and 5'-TCG TTG CTT GGT TCT CCT 10% heat-inactivated fetal bovine serum (FBS; HyClone, TG-3' (antisense); IL-6, 5'-AAC GAT GAT GCA CTT GCA Logan, UT, USA) and 1% antibiotic-antimycotic (Invitrogen, GA-3' (sense) and 5'-GAG CAT TGG AAA TTG GGG Grand Island, NY, USA). The RAW264.7 cells were maintained TA-3' (antisense); TNF-α were 5'-CAT CTT CTC AAA ATT by weekly passage, and the cells were utilized for experimen- CGA GTG ACA A-3' (sense) and 5'-TGG GAG TAG ACA tation at 60-80% confluence. Following pre-incubation of AGG TAC AAC CC-3' (antisense); and β-actin, 5'-TGT TTG the RAW264.7 cells for 4 h, 0-40 µg/ml of the extract were AGA CCT TCA ACA CC-3' (sense) and 5'-CGC TCA TTG added. Bay 11-7082 was used to inhibit the NF-κB pathway. CCG ATA GTG AT-3' (antisense). β-actin was included as an Bay 11-7082, also known as (E)-3-(4-methylphenylsulfonyl)- internal, housekeeping control gene.
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